Throughout this application various publications are referenced by Arabic numerals. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
21-Hydroxylase
The adrenal gland and the pituitary gland operate in a feedback loop to produce cortisol. The pituitary gland produces adrenocorticotrophic hormone (ACTH), which stimulates cortisol production by the adrenal gland. Cortisol causes suppression of ACTH production.
In the majority of cases of congenital adrenal hyperplasia, the production of cortisol by the adrenal cortex is impaired because of a defect in, or absence of, the gene for steroid 21-hydroxylase, an enzyme necessary for cortisol synthesis. Because of the failure to produce cortisol, unsuppressed production of ACTH causes the adrenal cortex to make excessive amounts of glucocorticoid related hormones. Some of these hormones act as androgens. As a result, an affected female will have masculinized genitalia at birth. It has been shown that small amounts of glucocorticoids, administered during pregnancy, can suppress fetal adrenal hormone production and prevent these genital abnormalities. In some cases aldosterone production is also impaired. As a result affected individuals develop the most severe form of disease, the salt wasting form of congenital adrenal hyperplasia [1].
The gene encoding 21-hydroxylase is on the short arm of chromosome 6, where a gene (CYP21B), and a pseudogene (CYP21A), alternate with the complement C4 genes [2, 3]. DNA Sequence analysis showed that CYP21B and CYP21A are highly homologous, but several deletions and mutations are present in the pseudogene which render it nonfunctional [4, 5]. Studies using oligonucleotide probes specific for CYP21A and CYP21B, Southern blot/restriction fragment length polymorphism (RFLP), PCR/RFLP or PCR/oligonucleotide probes and sequence analysis of these genes revealed that point mutations, gene deletion, and specific gene to pseudogene conversions are the abnormalities that most often affect the CYP21B alleles in patients with congenital adrenal hyperplasia [6-22].
Most of these methods require the use of molecular hybridization and some are too labor intensive for routine clinical diagnosis or for larer epidemiological studies. We designed and tested, on control human DNA and DNAs prepared from seven patients with congenital adrenal hyperplasia and their family members a method that rapidly and accurately differentiates between the CYP21B and CYP21A genes and conversion events. The method is based on PCR amplification and RFLP analysis of the amplified material; it does not require hybridization analysis, it allows a near perfect discrimination between normal and affected individuals for all of the catalogued abnormalities associated with disease, and it can be easily performed in any laboratory.
Human Papillomavirus
Over the last ten years the association between specific types of human papillomavirus (HPV) and cervical cancers and their precursors has been studied [23-30, 45, 46]. Over 65 different types of HPV have been described and more than 20 of these infect the male and female anogenital tract. Based on their associations with specific types of clinical lesions, the anogenital HPV types have been divided into three classes; low, medium and high oncogenic risk types of viruses [31]. The low oncogenic risk category includes such types as HPVs 6 and 11 which are commonly detected in condylomata acuminata but rarely associated with high-grade cervical intraepithelial neoplasia lesions (CIN) and cancer. The medium oncogenic risk category includes such types as 31, 35 and 45 which are often found in high grade CIN lesions but are only occasionally detected in invasive cancers. Finally, the high oncogenic risk category includes such types as HPVs 16, 18, and 33 which are often found in association with high-grade CIN lesions and cervical cancers.
Despite the potential clinical importance of differentiating between HPV types, current methods for detecting and typing HPV all suffer from serious limitations. Southern blots are considered to be the "gold standard" for HPV testing because of their high sensitivity and capacity to discriminate between the different types of HPV. However, Southern blot hybridization is too labor-intensive for routine clinical testing or for large-scale epidemiological studies. Dot blots are more rapid and easier to perform than Southern blots but are slightly less sensitive and require a large number of different hybridization steps in order to discriminate between individual HPV types. Analysis of the published sequences of human papillomaviruses by Manos and her colleagues [29] revealed a high degree of sequence conservation within the L1 open reading frame (ORF). This homology led to their devising a pair of degenerate "consensus primers" that could be used to amplify virtually any HPV DNA by the polymerase chain method (PCR). PCR with consensus primers allows for rapid detecting of virus sequences in clinical specimens. However, virus typing requires further analysis employing transfer of the reaction products to an immobilized support and multiple rounds of molecular hybridization with "type-specific" probes to discriminate between HPV types. In one recent publication more than 30 type-specific probes were required to discriminate between 17 types of HPV [32].
Detection And Species Identification Of Mycobacteria By PCR-RFLP Analysis
Isolation and identification of mycobacteria species takes several weeks when culturing is required. An alternative method that requires less than 24 hours for identification is enzymatic amplification (PCR) [33] of mycobacterial DNA. However, speciation requires further analysis employing molecular hybridization with "species-specific" probes, analysis that tends to be cumbersome and time consuming. We have devised a rapid and easy method for determining the species of mycobacterium from amplified mycobacterial DNA.